The Sabin vaccine strain of type 3 poliovirus (PS3) is temperature sensitive (growing at 37 but not at 39 degrees Centigrade) whereas its parent the Leon (PL3) strain is not. The ts phenotype of the Sabin strain has been shown to be due to a substitution of phe (PS3) for ser (PL3) at position 91 of the capsid protein VP3. The temperature sensitivity of PS3 is known to contribute to its attenuation and may be associated with limitations in the physical stability of vaccine stocks upon storage. PS3 has been shown to revert to a neurovirulent phenotype with a low but significant frequency upon replication in human vaccines. A number of isolates from vaccine associated cases of poliomyelitis have been found to have reverted to a ts(r) phenotype. Sequencing studies have shown that in a majority of isolates the reversion to ts(r) involves second site mutations (i.e. they still contain phe at residue 91 of VP3). The sequence changes associated with the ts phenotype and the ts to ts(r) reversion have been located in the structure of the Mahoney stain of type 1 poliovirus and were found to cluster in the interface between fivefold related protomers. We propose to use a combination of biological characterization, crystallographic studies, and molecular modeling methods to investigate the structural basis for temperature sensitivity in PS3. Temperature shift studies will be used to define the stage of the virus life cycle (attachment, uncoating, assembly) which the ts lesion affects. Crystallographic and molecular modeling (direct model building and energy minimization) studies of ts(r) revertant strains will be used to define the structural consequences of sequence changes associated with ts/ts(r) transitions. The structures derived from direct crystallographic studies will be compared with those derived from molecular modeling to asses the ability of modeling to predict the structural consequences of limited sequence changes. Energy calculations will be used to quantitate the effect of sequence/structure changes on virion stability. These results will be assessed for their ability to predict the phenotype of the relevant stains and for possible implications for the manipulation of the ts phenotype of PS3.